This research will develop techniques for using cell-free enzymes as catalysts in organic synthesis. A previously tested ATP regeneration scheme will be used in conjunction with ATP requiring ligases in synthesis of NAD(P), phosphoribosyl pyrophosphate, and ATP-gamma-S. An ATP-driven reaction sequence will regenerate and utilize UDP-glucose. Reconstituted cytochrome P450 monooxygenase systems will be used to prepare prenenolone and 1,25-dihydroxy-vitamin-D3. Enatioselective enzymatic syntheses of L-glycerol phosphate and of alpha-hydroxy carboxylic acids will be developed. The potential utility of semisynthetic enzymes prepared by site-specific modification of proteins with non-enzymatic catalytic groups will be investigated. The research will emphasize the development of general, rational procedures for enzyme stabilization, cofactor regeneration, and enzyme utilization in synthesis.